Aspartate is associated with the synaptic vesicles of certain excitatory hippocampal pathways and is released by hippocampal slices and synaptosomes in a calcium-dependent manner. Preliminary release studies and information available from other experimental approaches allowed construction of a neuropeptide-like working model for aspartate release and function that includes accumulation into a distinct vesicle population, vesicle fusion with the plasma membrane predominantly outside the active zone, and diffuse activation of NMDA receptors. Aspartate, rather than glutamate, may produce some of the global or extrasynaptic effects of NMDA receptor activation (apoptosis, regulation of neurogenesis, and regulation of axonal/dendritic growth). The proposed release studies will utilize rat hippocampal synaptosomes. A method will be developed to introduce polypeptides and proteins into the synaptosomes by an electroporation technique that does not disrupt glutamate/aspartate release significantly. Then, to discriminate among the possible mechanisms of aspartate release suggested by previous work, the electroporation method will be used to introduce complexin inhibitory peptide, NSF inhibitory peptide, botulinum toxin A light chain, and botulinum toxin B light chain into the synaptosomes. Whole cell patch clamp recordings in organotypic hippocampal slice cultures will test the hypothesis that aspartate release plays a role in excitatory synaptic transmission under certain conditions. These studies will exploit several apparent differences between aspartate and glutamate in their release mechanisms and receptor actions, including release of aspartate from the Schaffer collateral-commissural pathway but not from the hippocampal mossy fibers, increased aspartate under low glucose conditions, increased aspartate release and reduced glutamate release during repetitive activity, insensitivity of aspartate release to Clostridial toxins, lower sensitivity of aspartate release to a blocker of P/Q-type calcium channels, and agonist activity of aspartate upon NMDA but not upon AMPA receptors. The proposed studies will evaluate a potential role for aspartate as a signaling molecule in the brain. They require, in part, the development of new experimental approaches and involve some risk of failure. For these reasons, the Exploratory/Developmental Grant mechanism of support seems appropriate. This project will eventually shed light on developmental and neurodegenerative processes, including the pathophysiology and pathology associated with epileptic seizures and hypoglycemia.